EP3526321A1 - Utilisation d'agents tonifiants pour augmenter le rendement de virus adéno-associés recombinés - Google Patents

Utilisation d'agents tonifiants pour augmenter le rendement de virus adéno-associés recombinés

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Publication number
EP3526321A1
EP3526321A1 EP17859455.2A EP17859455A EP3526321A1 EP 3526321 A1 EP3526321 A1 EP 3526321A1 EP 17859455 A EP17859455 A EP 17859455A EP 3526321 A1 EP3526321 A1 EP 3526321A1
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EP
European Patent Office
Prior art keywords
cell culture
osmolality
culture medium
medium
cell
Prior art date
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EP17859455.2A
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German (de)
English (en)
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EP3526321A4 (fr
Inventor
Ying JING
Jan PANTELI
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Ultragenyx Pharmaceutical Inc
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Dimension Therapeutics Inc
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Publication of EP3526321A1 publication Critical patent/EP3526321A1/fr
Publication of EP3526321A4 publication Critical patent/EP3526321A4/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14151—Methods of production or purification of viral material
    • C12N2750/14152—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles

Definitions

  • the invention relates generally to methods for enhancing recombinant adeno-associated virus vector (rAAV) yield, and, more particularly, the invention relates to the use of tonicifying agents to enhance rAAV yield.
  • rAAV adeno-associated virus vector
  • Adeno-associated virus is a non-pathogenic, replication-defective parvovirus.
  • Recombinant AAV vectors have many unique features that make them attractive as vectors for gene therapy.
  • rAAV vectors can deliver therapeutic genes to dividing and nondividing cells, and these genes can persist for extended periods without integrating into the genome of the targeted cell.
  • cell culture additives such as metals, trace supplements, salts, and others.
  • rAAV are often produced by means of a helper virus, such as adenovirus (AV).
  • helper virus such as adenovirus (AV).
  • AV adenovirus
  • any helper virus is a contaminant that must be removed before the rAAV can be used in therapeutic applications. There is therefore also a need in the art to produce rAAV with as little contaminating helper virus as possible.
  • the invention is based, in part, upon the discovery that a host cell used in the production of recombinant adeno-associated virus vectors (rAAV) will produce increased amounts of rAAV and decreased amounts of helper virus when the osmolality of the cell culture media is increased via the addition of an ionic tonicifying agent, such as NaCl.
  • rAAV recombinant adeno-associated virus vectors
  • the invention is also based, in part, on the discovery that a host cell used in the production of rAAV will produce decreased amount of helper virus when the osmolality of the cell culture media is increased via the addition of a non-ionic tonicifying agent such as sucrose.
  • the invention provides a method for producing a rAAV comprising incubating a host cell in a culture media with increase osmolality.
  • the invention provides a method for decreasing the amount of helper virus produced by a rAAV-producing host cell, comprising incubating the rAAV-producing host cell in cell culture media with increased osmolality due to supplementation with a non-ionic tonicifying agent such as sucrose.
  • the invention provides a method for increasing the production of rAAV and decreasing the production of helper virus produced by a host cell, comprising incubating the host cell in cell culture media with increased osmolality due to supplementation with an ionic tonicifying agent such as NaCl.
  • the invention relates to cell culture systems comprising a host cell capable of producing both rAAV and helper virus, and a cell culture media with increased osmolality due to supplementation with a tonicifying agent.
  • the tonicifying agent is ionic. It is contemplated that the tonicifying agent may be any ionic tonicifying agent compatible with mammalian or insect cell culture media. Exemplary ionic tonicifying agents include NaCl, KC1, NaN0 3 , NaHC0 3 , Na 2 S0 4 , Na 2 HP0 4 , NaH 2 P0 4 , NaN0 3 , KN0 3 , K 2 S0 4 , K 2 HP0 4 , KH 2 P0 4 , or KN0 3 . In one embodiment, the tonicifying agent is NaCl.
  • the tonicifying agent is NaCl
  • it may be present at a concentration of at least 4.5 g/L (77.0 mM). It is further contemplated that NaCl may be present at a concentration of at least 6.5 g/L (111.2 mM), or at least 7.5 g/L (128.3 mM). It will be understood that other ionic tonicifying agents can be substituted for NaCl by substituting an equal osmolar amount of the alternative salt.
  • the tonicifying agent is non-ionic. It is contemplated that the tonicifying agent may be any non-ionic tonicifying agent compatible with mammalian or insect cell culture media.
  • the non-ionic tonicifying agents may be a sugar, including disaccharides and monosaccharides, such as sucrose, fructose, glucose, galactose, mannose, maltose, and trehalose.
  • the tonicifying agent is sucrose. It is contemplated that, where the tonicifying agent is sucrose, it may be present at a concentration of at least 6.8 g/L (19.9 mM).
  • sucrose may be present at a concentration of at least 13.7 g/L (40.0 mM), 29.4 g/L (85.9 mM), or 41.1 g/L (120.0 mM). It will be understood that other non-ionic tonicifying agents can be substituted for sucrose by substituting an equal molar amount of the alternative non-ionic tonicifying agent.
  • the osmolality of the culture media when measured at the start of incubation of the host cell, will be 360 mOsm/kg or higher, 375 mOsm/kg or higher, or 400 mOsm/kg or higher.
  • the osmolality of the culture medium is sufficient to produce at least a 20% reduction in total helper virus production, relative to that produced by a host cell in a cell culture medium at 266 mOsm/kg. It is further contemplated that the reduction in total helper virus production may be 30%, 40%, or 50%.
  • the concentration of ionic tonicifying agent is sufficient to produce at least a 50% increase in total rAAV production, relative to that produced by a host cell in a cell culture medium at 266 mOsm/kg. It is further contemplated that the increase in total rAAV production may be 100%, 150%, or 200%. In one embodiment, the period of time the host cell incubated in the cell culture medium with increased osmolality is for at least 2 days. It is further contemplated that the incubation may be at least 3 days or about 4 days.
  • the host cell may be a mammalian cell, for example, a HeLa, HEK293, COS, A549, BHK, or Vero cell. It is also contemplated that the host cell may be an insect cell, for example, a Sf9, Sf-21, Tn-368, or BTI-Tn-5Bl-4 (High-Five) cell. In one embodiment, the host cell is a HeLa cell. It is contemplated that the host cell may comprise a heterologous nucleotide sequence flanked by AAV inverted terminal repeats, rep and cap genes, or helper virus genes.
  • the host cell comprises a heterologous nucleotide sequence flanked by AAV inverted terminal repeats, rep and cap genes, and helper virus genes. In one embodiment, the host cell comprises one or more AAV genes stably integrated into the host cell's genome.
  • the helper virus may be any virus capable of allowing AAV contained in a host cell to enter the infections phase, for example adenovirus, herpes simplex virus, papilloma virus, or baculovirus.
  • the helper virus is adenovirus subtype 5 (Ad5).
  • the host cell will be capable of producing both rAAV and helper virus.
  • a host cell is capable of producing rAAV or AV if, in the absence of intervention and given appropriate culture conditions, the cell will produce viral particles, whether or not the particles are released into the cell culture media.
  • a host cell may be capable of producing a virus because it was infected by a live virus, or because it was transfected with viral genes that may exist in the cell transiently, for example, on a plasmid or other extrachromosomal body, or be permanently integrated into the host cell genome.
  • host cells transiently transfected with one or more plasmids containing AAV inverted terminal repeats, rep and cap genes, and infected by live helper virus may be considered capable of producing both rAAV and helper virus. It is further contemplated that host cells containing AAV inverted terminal repeats, rep and cap genes integrated in the host cell's chromosomes and infected with a live helper virus will be considered capable of producing both rAAV and helper virus.
  • the host cell is inoculated with non-replicating helper virus.
  • the host cell is capable of producing rAAV but not capable of producing helper virus, and thus the beneficial effects of increased osmolality on helper virus production will not occur, however the beneficial effect of increased rAAV production will still be realized.
  • the invention provides a rAAV produced by any of the contemplated methods, a composition comprising a rAAV produced by any of the contemplated methods, or a cell culture system comprising a host cell capable of producing both rAAV and helper virus and cell culture media with an osmolality of 360 mOsm/kg or higher.
  • FIG. 1A depicts the effect of the addition of the ionic tonicifying agent NaCl on the production of extracellular rAAV and Ad5 in a HeLa producer cell line at 4 days post inoculation and infection. rAAV and Ad5 production was quantified by qPCR. Each value represents the mean of two independent experiments.
  • FIG. IB depicts the effect of the addition of the ionic tonicifying agent NaCl on the production of total rAAV and Ad5 in a HeLa producer cell line at 4 days post inoculation and infection. rAAV and Ad5 production was quantified by qPCR. Each value represents the mean of two independent experiments.
  • FIG. 2A depicts the effect of the addition of the non-ionic tonicifying agent sucrose on the production of extracellular rAAV and Ad5 in a HeLa producer cell line at 4 days post inoculation and infection.
  • rAAV and Ad5 production was quantified by qPCR. Each value represents the mean of two independent experiments.
  • FIG. 2B depicts the effect of the addition of the non-ionic tonicifying agent sucrose on the production of total rAAV and Ad5 in a HeLa producer cell line at 4 days post inoculation and infection.
  • rAAV and Ad5 production was quantified by qPCR. Each value represents the mean of two independent experiments.
  • the invention is based, in part, upon the discovery that the production of rAAV and helper virus in a host cell can be optimized by increasing the osmolality of the culture media through the use of a tonicifying agent, such as NaCl or sucrose.
  • a tonicifying agent such as NaCl or sucrose.
  • the invention provides a method for producing rAAV using a helper virus, the method comprising incubating a host cell capable of producing both rAAV and helper virus in a cell culture medium containing one or more tonicifying agents and having an osmolality of 360 mOsm/kg or higher at the start of the incubation period.
  • the invention provides a method for decreasing the amount of helper virus produced during the production of rAAV by incubating a host cell in a cell culture medium containing one or more tonicifying agents and having an osmolality of 360 mOsm/kg or higher at the start of the incubation period.
  • the invention provides a method for increasing the amount of rAAV produced by a host cell while simultaneously decreasing the amount of helper virus produced by a host cell, by incubating a host cell in a cell culture medium containing one or more tonicifying agents and having an osmolality of 360 mOsm/kg or higher at the start of the incubation period.
  • Adeno-associated virus is a small, nonenveloped icosahedral virus of the genus Dependoparvovirus and family Parvovirus.
  • AAV has a single-stranded linear DNA genome of approximately 4.7 kb.
  • AAV includes numerous serologically distinguishable types including serotypes AAV-1 to AAV-12, as well as more than 100 serotypes from nonhuman primates (See, e.g. , Srivastava, J. Cell Biochem, 105(1): 17-24 (2008), and Gao et al, J. Virol, 78(12), 6381-6388 (2004)). Any AAV type may be used in the methods of the present invention.
  • AAV is capable of infecting both dividing and quiescent cells of several tissue types, with different AAV serotypes exhibiting different tissue tropism.
  • AAV is non-autonomously replicating, and has a life cycle with a latent phase and an infectious phase.
  • the latent phase after a cell is infected with an AAV, the AAV site-specifically integrates into the host's genome as a provirus.
  • the infectious phase does not occur unless the cell is also infected with a helper virus (for example, adenovirus (AV) or herpes simplex virus (HSV)), which allows the AAV to replicate.
  • helper virus for example, adenovirus (AV) or herpes simplex virus (HSV)
  • the wild-type AAV genome contains two 145 nucleotide inverted terminal repeats
  • ITRs which contain signal sequences directing AAV replication, genome encapsidation and integration.
  • ITRs three AAV promoters, p5, pi 9, and p40, drive expression of two open reading frames encoding rep and cap genes.
  • Rep proteins are responsible for genomic replication.
  • the Cap gene is expressed from the p40 promoter, and encodes three capsid proteins (VP1, VP2, and VP3) which are splice variants of the cap gene. These proteins form the capsid of the AAV particle.
  • the cw-acting signals for replication, encapsidation, and integration are contained within the ITRs, some or all of the 4.3 kb internal genome may be replaced with foreign DNA, for example, an expression cassette for an exogenous protein of interest.
  • the rep and cap proteins are provided in trans on, for example, a plasmid.
  • a host cell line permissive of AAV replication must express the rep and cap genes, the ITR-flanked expression cassette, and helper functions provided by a helper virus, for example AV genes Ela, Elb55K, E2a, E4orf6, and VA (Weitzman et al , Adeno- associated virus biology.
  • Adeno-Associated Virus Methods and Protocols, pp. 1-23, 2011).
  • Production of AAV vector can also result in the production of helper virus particles, which must be removed or inactivated prior to use of the AAV vector.
  • Numerous cell types are suitable for producing AAV vectors, including HEK293 cells, COS cells, HeLa cells, BHK cells, Vero cells, and A549 cells, as well as insect cells, including Sf9, Sf-21, Tn-368, and BTI- Tn-5Bl-4 (High-Five) cells (See e.g. U.S. Pat. Nos. 6,156,303, 5,387,484, 5,741,683,
  • AAV vectors are typically produced in these cell types by one plasmid containing the ITR-flanked expression cassette, and one or more additional plasmids providing the additional AAV and helper virus genes.
  • AAV of any serotype may be used in the present invention.
  • any AV type may be used, and a person of skill in the art will be able to identify AAV and AV types suitable for the production of their desired recombinant AAV vector (rAAV).
  • AAV and AV particles may be purified, for example by affinity chromatography, iodixonal gradient, or CsCl gradient.
  • the genome of wild-type AAV is single-stranded DNA and is approximately 4.7 kb.
  • AAV vectors may have single-stranded genomes that are 4.7 kb in size, or are larger or smaller than 4.7 kb, including oversized genomes that are as large as 5.2 kb, or as small as 3.0 kb. Further, vector genomes may be substantially self-complementary, so that within the virus the genome is substantially double stranded.
  • AAV vectors containing genomes of all types are suitable for use in the method of the instant invention.
  • helper viruses include Adenovirus (AV), including recombinant AV, and herpes simplex virus (HSV), including recombinant HSV.
  • AV Adenovirus
  • HSV herpes simplex virus
  • Systems also exist for producing AAV in insect cells using baculovirus, including recombinant baculovirus.
  • papilloma viruses may provide a helper function for AAV (see, e.g. , Hermonat et al , Molecular Therapy 9, S289-S290 (2004)), and suitable papilloma viruses may be used in the methods of the instant invention.
  • Helper viruses include any virus capable of creating an allowing AAV replication.
  • AV is a nonenveloped nuclear DNA virus with a double-stranded DNA genome of approximately 36 kb.
  • AV is capable of rescuing latent AAV in a cell, by providing Ela, Elb55K, E2a, E4orf6, and VA genes, allowing AAV replication and encapsidation.
  • HSV is a family of viruses that have a relatively large double-stranded linear DNA genome encapsidated in an icosahedral capsid, which is wrapped in a lipid bilayer envelope. HSV are infectious and highly transmissible.
  • HSV-1 replication proteins were identified as necessary for AAV replication: the helicase/primase complex (UL5, UL8, and UL52) and the DNA binding protein ICP8 encoded by the UL29 gene, with other proteins enhancing the helper function.
  • the present invention comprises the production of a recombinant adeno-associated virus vector (rAAV) from a host cell, using any suitable method known in the art.
  • the term "host cell” refers to any cell or cells capable of producing a rAAV.
  • the host cell is a mammalian cell, for example, a HeLa cell, COS cell, HEK293 cell, A549 cell, BHK cell, or Vero cell.
  • the host cell is an insect cell, for example, a Sf9 cell, Sf-21 cell, Tn-368 cell, or BTI-Tn-5Bl-4 (High-Five) cell.
  • the terms "cell” or “cell line” are understood to include modified or engineered variants of the indicated cell or cell line.
  • the host cell must be provided with AAV inverted terminal repeats (ITRs) (which may, for example, flank a heterologous nucleotide sequence of interest), AAV rep and cap gene functions, as well as additional helper functions.
  • ITRs AAV inverted terminal repeats
  • additional helper functions can be provided by, for example, an adenovirus (AV) infection, by a plasmid that carries all of the required AV helper function genes, or by other viruses such as HSV or baculovirus.
  • the host cell is a producer cell comprising AAV rep and cap gene functions and a rAAV vector genome.
  • the host cell is a packaging cell comprising AAV rep and cap gene functions, which at the time of production is provided a rAAV vector genome by a separate recombinant virus.
  • rAAV production methods suitable for use with the methods of the current invention include those disclosed in Clark et al , Human Gene Therapy 6: 1329-1341 (1995), Martin et al , Human Gene Therapy Methods 24:253-269 (2013), Thome et al, Human Gene Therapy 20:707-714 (2009), Fraser Wright, Human Gene Therapy 20:698-706 (2009), and Virag et al , Human Gene Therapy 20:807-817 (2009). 3. Tonicifying Agents
  • any cell culture medium appropriate for propagation of the host cell may be used in the instant invention.
  • suitable cell culture media include minimum essential medium (MEM) such as Eagle's culture medium, Dulbecco's modified Eagle's medium (DMEM), minimum essential medium alpha (MEM-alpha), mesenchymal cell basal medium (MSCBM), Ham's F-12 medium and Ham's F-10 medium, DMEM/F12 medium, William's medium E, RPMI-1640 medium, MCDB medium, medium 199, Fisher's medium, Iscove's modified Dulbecco's medium (IMDM), Leibovitz's L-15 medium, and McCoy's modified medium.
  • MEM minimum essential medium
  • DMEM Dulbecco's modified Eagle's medium
  • MSCBM mesenchymal cell basal medium
  • MSCBM mesenchymal cell basal medium
  • Ham's F-12 medium Ham's F-10 medium
  • DMEM/F12 medium William's medium E
  • Osmolality is a measure of the number of dissolved particles per kilogram of solvent.
  • the osmolality of a solution can be measure by freezing point depression or vapor pressure depression.
  • a tonicifying agent is any agent capable of increasing the osmolality of a solution without substantially adversely affecting other important attributes of the solution. Appropriate tonicifying agents will thus vary depending on the nature and purpose of the solution. In the case of cell culture media, an appropriate tonicifying agent is one that is non-toxic, and will not substantially alter the pH, buffering capacity, and nutrient density of the media.
  • Ionic tonicifying agents are those that dissociate into ions, for example, salts such as NaCl, KC1, NaN0 3 , NaHC0 3 , Na 2 S0 4 , Na 2 HP0 4 , NaH 2 P0 4 , NaN0 3 , KN0 3 , K 2 S0 4 , K 2 HP0 4 , KH 2 P0 4 , and K 0 3 .
  • Non-ionic tonicifying agents are those that do not dissociate into ions, for example, sugars such as sucrose, fructose, glucose, galactose, mannose, maltose, and trehalose.
  • host cells are cultured in suspension culture.
  • metabolically active cells will acidify the cell culture medium in which they are grown, and the pH of the culture medium must be periodically adjusted via the addition of sterile Na 2 C0 3 or NaOH to return the pH to optimal levels. It will be apparent the addition of Na 2 C0 3 or NaOH, as well as the metabolic activity of the cells in culture will increase the osmolality of the culture media. For this reason, the increased osmolality of the instant invention is the lowest osmolality to which the cells are exposed, and the osmolality increases over the duration of culture.
  • the rAAV particles are harvested and/or purified from the host cell that has been incubated in cell culture media containing one or more tonicifying agents and having an osmolality of 360 mOsm/kg or higher at the start of the incubation period.
  • rAAV particles may be obtained from host cells by lysing the cells. Lysis of host cells can be accomplished by methods that chemically or enzymatically treat the cells in order to release infections viral particles. These methods include the use of nucleases such as benzonase or DNAse, proteases such as trypsin, or detergents or surfactants.
  • total rAAV refers to the total rAAV produced by a host cell
  • secreted rAAV or "extracellular rAAV” refers to rAAV that can be can be harvested from a host cell without a cell lysis step.
  • rAAV particles After harvesting rAAV particles, it may be necessary to purify the sample containing rAAV to remove, for example, the cellular debris resulting from cell lysis.
  • Methods of minimal purification of AAV particles are known in the art. Two exemplary purification methods are Cesium chloride (CsCl)- and iodixanol-based density gradient purification. Both methods are described in Strobel et al, Human Gene Therapy Methods., 26(4): 147-157 (2015).
  • Minimal purification can also be accomplished using affinity chromatography using, for example AVB Sepharose affinity resin (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).
  • rAAV particles can be quantified using a number of methods, however, including quantitative polymerase chain reaction (qPCR) (Clark et al, Hum. Gene Ther. 10, 1031-1039 (1999)) or dot-blot hybridization (Samulski et al, J. Virol. 63, 3822-3828 (1989)), or by optical density of highly purified vector preparations (Sommer et al, Mol. Ther. 7, 122- 128 (2003)).
  • qPCR quantitative polymerase chain reaction
  • DNase-resistant particles can be quantified by real-time quantitative polymerase chain reaction (qPCR) (DRP-qPCR) in a thermocycler (for example, an iCycler iQ 96-well block format thermocycler (Bio-Rad, Hercules, CA)).
  • qPCR quantitative polymerase chain reaction
  • thermocycler for example, an iCycler iQ 96-well block format thermocycler (Bio-Rad, Hercules, CA)
  • samples containing rAAV particles are incubated in the presence of DNase I (100 U/ml; Promega, Madison, WI) at 37°C for 60 min, followed by proteinase K (Invitrogen, Carlsbad, CA) digestion (10 U/ml) at 50°C for 60 min, and then denatured at 95°C for 30 min.
  • the primer- probe set used should be specific to a non-native portion of the rAAV vector genome, for example, the poly(A) sequence of the protein of interest.
  • the PCR product can be amplified using any appropriate set of cycling parameters, based on the length and composition of the primers, probe, and amplified sequence. Alternative protocols are disclosed in, for example, Lock et al, Human Gene Therapy Methods 25(2): 115-125 (2014).
  • the infectivity of rAAV particles can be determined using a TCID50 (tissue culture infectious dose at 50%) assay, as described for example in Zhen et al, Human Gene Therapy 15:709-715 (2004).
  • TCID50 tissue culture infectious dose at 50%
  • rAAV vector particles are serially diluted and used to co- infect a Rep/Cap-expressing cell line along with AV particles in 96-well plates. 48 hours postinfection, total cellular DNA from infected and control wells is extracted. rAAV vector replication is then measured using qPCR with transgene-specific probe and primers.
  • TCID50 infectivity per milliliter (TCID50/ml) is calculated with the Karber equation, using the ratios of wells positive for AAV at 10-fold serial dilutions.
  • a HeLa producer cell line derived from the HeLa S3 parental cell line was used to produce rAAV in this experiment.
  • the vector sequence, the AAV rep and cap genes and a selectable marker gene was transfected into HeLa S3 cells, and a stable integrant was selected.
  • the cells were cultured in protein free, chemically defined cell culture medium with a 10% fraction of culture medium carried over through inoculation.
  • Culture media were supplemented with NaCl to a final osmolality either 266, 342, or 370 mOsmo/Kg in 250 mL shake flasks with a starting volume of 100 mL and an initial cell density of 1 ⁇ 10 6 cells/mL.
  • Cells were maintained at 37°C and 5% CO2 for 4 days with 10% fraction of feed for all conditions.
  • the cultures were sampled daily to monitor cell growth and metabolites and the pH was adjusted equally across flasks as needed using 1M Na2CC>3. At the end of 4 days in culture, total and extracellular yield of rAAV and Ad5 was determined.
  • Extracellular yield was determined by removing samples of cell culture media containing suspended cells from each shake flask at the end of 4 days, filtering with 0.2 ⁇ PES (polyethersulfone) syringe filter and quantifying the rAAV and Ad5 genomes in each sample using quantitative real-time PCR.
  • PES polyethersulfone
  • Total yield was determined by removing samples of cell culture media containing suspended cells from each shake flask at the end of 4 days, lysing cells with detergent, filtering with 0.2 ⁇ PES (polyethersulfone) syringe filter, and quantifying the rAAV and Ad5 genomes in each sample using quantitative real-time PCR.
  • PES polyethersulfone
  • Results of this experiment are presented in FIG. 1 A and IB.
  • increasing the osmolality of culture media by adding an ionic tonicifying agent, NaCl resulted in an increase in the production of extracellular rAAV, with no significant change in the production of extracellular Ad5.
  • increasing the osmolality of culture media by adding an ionic tonicifying agent, NaCl resulted in an increase in the production of total rAAV, together with a decrease in production of total Ad5.
  • Cells were cultured as described in Example 1, with the exception that media were supplemented with the non-ionic tonicifying agent sucrose to a final osmolality of 266, 286, 305, 353 or 390 mOsmo/Kg and no additional NaCl was added.
  • Results from this experiment are presented in FIG. 2A and 2B.
  • increasing the osmolality of culture media by adding a non-ionic tonicifying agent, sucrose resulted in an increase in extracellular rAAV production and a decrease in extracellular Ad5 production.
  • increasing the osmolality of culture media by adding a non-ionic tonicifying agent, sucrose resulted in no change in total rAAV production and a decrease in total Ad5 production.
  • a method for producing a recombinant adeno-associated virus vector (rAAV) using a helper virus comprising incubating a host cell capable of producing rAAV in the presence of helper virus for an incubation period in a cell culture medium containing helper virus and one or more tonicifying agents and having an osmolality of 360 mOsm/kg or higher at the start of the incubation period.
  • rAAV a recombinant adeno-associated virus vector
  • the host cell comprises a genome, said genome comprising one or more AAV genes stably integrated therein.
  • the cell culture medium has an osmolality at the start of the incubation period sufficient to produce a 40% reduction in total helper virus production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.
  • the cell culture medium has an osmolality at the start of the incubation period sufficient to produce at least a 50% increase in total rAAV production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.
  • the cell culture medium has an osmolality at the start of the incubation period sufficient to produce at least a 100% increase in total rAAV production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.
  • the cell culture medium has an osmolality at the start of the incubation period sufficient to produce at least a 150% increase in total rAAV production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.
  • At least one tonicifying agent is an ionic tonicifying agent.
  • at least one tonicifying agent is selected from the group comprising: NaCl, KC1, NaN0 3 , NaHC03, Na 2 S0 4 , Na 2 HP0 4 , NaH 2 P0 4 , NaN0 3 , KN0 3 , K 2 S0 4 , K 2 HP0 4 , KH 2 P0 4 , and KN0 3 .
  • one tonicifying agent is NaCl.
  • the cell culture medium contains an ionic tonicifying agent at a concentration sufficient to produce at least a 100% increase in total rAAV production and a 30% decrease in helper virus production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.
  • the cell culture medium contains an ionic tonicifying agent at a concentration sufficient to produce at least a 200% increase in total rAAV production and a 50% decrease in helper virus production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.
  • the tonicifying agent is selected from the group consisting of sucrose, fructose, glucose, galactose, mannose, maltose, and trehalose.
  • the tonicifying agent is sucrose.
  • cell culture medium is selected from the group consisting of MEM, DMEM, RPMI, Ham's F-12 medium, Leibovitz's L-15 medium, and mixtures thereof, said medium being supplemented with one or more tonicifying agents.
  • the cell culture medium consists essentially of DMEM supplemented with one or more tonicifying agents.
  • the host cell is a mammalian cell.
  • the host cell is selected from the group consisting of HeLa, HEK293, COS, A549, BHK, and Vero cells.
  • helper virus is selected from the group consisting of adenovirus, herpes virus, baculovirus, and recombinant forms of any of the foregoing viruses.
  • helper virus is an adenovirus (AV).
  • the host cell comprises a heterologous nucleotide sequence flanked by AAV inverted terminal repeats, rep and cap genes, and helper virus genes.
  • a cell culture system comprising: a. a host cell capable of producing rAAV; b. a helper virus; and c. a cell culture medium with an osmolality of 360 mOsm/kg or higher when measured immediately after the host cell is introduced into the cell culture medium.
  • a. a host cell capable of producing rAAV a helper virus
  • 55 The cell culture system of claim 54, wherein the cell culture medium has an osmolality of 375 mOsm/kg or higher.
  • cell culture system of claim 72 wherein the cell culture medium contains an ionic tonicifying agent at a concentration sufficient to produce at least a 100% increase in total rAAV production and a 30% decrease in helper virus production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.
  • the cell culture system of claim 72 wherein the cell culture medium contains an ionic tonicifying agent at a concentration sufficient to produce at least a 150% increase in total rAAV production and a 40% decrease in helper virus production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.
  • the cell culture medium contains an ionic tonicifying agent at a concentration sufficient to produce at least a 200% increase in total rAAV production and a 50% decrease in helper virus production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.
  • cell culture system of claim 78 wherein the tonicifying agent is selected from the group consisting of sucrose, fructose, glucose, galactose, mannose, maltose, and trehalose.
  • the cell culture system of claim 79 wherein the tonicifying agent is sucrose.
  • the concentration of sucrose in the cell culture medium is 6.8 g/L or higher.
  • cell culture medium is selected from the group consisting of MEM, DMEM, RPMI, Ham's F-12 medium, Leibovitz's L-15 medium, and mixtures thereof, said medium being supplemented with one or more tonicifying agents.
  • the cell culture system of claim 89 wherein the host cell is selected from the group consisting of HeLa, HEK293, COS, A549, BHK, and Vero cells.
  • helper virus is selected from the group consisting of adenovirus, herpes virus, baculovirus, and recombinant forms of any of the foregoing viruses.

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Abstract

L'invention concerne des procédés de production de vecteurs de virus adéno-associés recombinés (rAAV), comprenant la culture de cellules productrices dans des milieux présentant une osmolalité accrue. L'invention concerne également des procédés pour diminuer la production d'un virus auxiliaire par une cellule productrice de rAAV, comprenant la culture de la cellule productrice dans des milieux avec une osmolalité accrue.
EP17859455.2A 2016-10-14 2017-10-13 Utilisation d'agents tonifiants pour augmenter le rendement de virus adéno-associés recombinés Pending EP3526321A4 (fr)

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